The Nitrated Proteome in Heart Mitochondria of the db/db Mouse Model: Characterization of Nitrated Tyrosine Residues in SCOT Yuan Wang, Fuli Peng, Wei Tong, Haidan Sun, Ningzhi Xu, and Siqi Liu* Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, and Beijing Proteomics Institute, Beijing, China Received September 17, 2009
Abstract: A proteomic strategy combining 2DE, Western blot, and mass spectrometry was implemented to survey the status of tyrosine nitration in mouse heart mitochondria. Compared to normal mice, nitrated proteins in the heart mitochondria of the db/db mouse model were significantly augmented due to diabetic development. A total of 18 proteins were identified as the nitration targets. Of the nitrated proteins, succinyl-CoA:3-oxoacid CoAtransferase (SCOT) is a key enzyme involved in ketolysis and has yet to be explored how its catalysis is affected by nitration. We therefore initiated a systematic investigation toward the nitrated site(s) and the corresponding changes of SCOT catalysis. To monitor modification kinetics and nitrated residue(s), recombinant SCOT was incubated with peroxynitrite followed by examination of nitration development as well as catalytic activity changes. The nitration of recombinant SCOT steadily increased in response to increasing concentrations of peroxynitrite, while its catalysis was gradually attenuated. The nitrated sites of modified SCOT were further identified by LC-ESIMS/MS. The MS/MS spectra indicated a +45 mass unit ion shift from [M + H]+ m/z at Tyr4 and Tyr76. Through site-directed mutagenesis, we found that mutation of tyrosine residues at Tyr4 or Tyr76 did not only significantly protect SCOT from peroxynitrite modification, but it also dramatically prevented loss of enzymatic activity. Taken together, these results indicate that the two tyrosine residues of SCOT are the priority sites attacked by NO, and their nitration status is a causal factor leading to inhibition of SCOT catalysis. Keywords: Diabetic Model • Mitochondria • Tyrosine Nitration • 2DE-Western Blot • 3NT • SCOT • Peroxynitrite • LC-ESI-MS/MS
Introduction Diabetes is a life-threatening disease that affects millions worldwide. There is increasing evidence that the production of nitric oxide (NO) is enhanced in diabetic tissues, which may result from activation of nitric oxide synthase (NOS) and/or * To whom correspondence should be addressed. Siqi Liu, Ph.D, Center of Proteomic Analysis, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing Airport Industrial Zone B-6, Beijing 101300, China. E-mail:
[email protected].
4254 Journal of Proteome Research 2010, 9, 4254–4263 Published on Web 06/09/2010
augmentation of reactive oxygen species (ROS) during chronic development.1,2 NO is a short-lived and highly reactive molecule.3 With the ability to easily cross membranes, NO is able to diffuse distances of more than several hundred micrometers, and plays an important signaling role in many tissues leading to modulation of physiological and cellular processes. One of major biochemical functions derived from NO is modification of amino acid residues in proteins. Peroxynitrite (ONOO-), a reactive form of NO in vivo, can modify cysteine, tryptophan, methionine, and tyrosine residues in proteins.4 As identified, increased nitrotyrosine and nitrotryptophan were found in rat heart, mainly in mitochondria, of diabetes and aging models.5,6 The nitration of mitochondrial protein consequently triggers mitochondrial dysfunction in diabetes.7-11 Tyrosine nitration of protein and its correlations with diseases are reported in several documents.1,12-16 Most studies related with the nitrated proteome are restricted to profiling the nitrated proteins, and the functions of nitrated proteins have yet to be deeply explored. For instance, there are many mitochondrial proteins sensitive to nitration stress, yet the nitration sites in these proteins have not been identified and the functional changes of these proteins in response to nitration stress are poorly characterized.17-19 Thus, combining a proteomic survey with functional inquiry is a promising strategy in the field. The ketoacidosis found in most diabetic patients is likely caused by reduction in the enzymatic activity of succinyl-CoA: 3-ketoacidCoAtransferase(SCOT,EC2.8.3.5)inmitochondria.20-22 Murad’s group first investigated the nitration of mitochondrial proteins in streptozotocin-treated rats. In their study, catalytic activity of rat heart SCOT was reduced 24% and 39% in the animals with streptozotocin administration for 4 and 8 weeks, respectively. More importantly, the decrease in SCOT catalytic activity was accompanied by accumulation of nitrotyrosine in SCOT protein.5 Molecular details such as which amino acid residues were nitrated and how SCOT activity correlated with the modification(s) were not fully elucidated in these studies. To identify the nitration site(s) in SCOT, Rebrin et al. used the 3NT antibody to enrich modified protein and then employed mass spectrometry to identify the nitrated residue(s).6 Conflicting data were reported in which SCOT nitration did not reduce its activity, but rather stimulated it approximately 10-fold, while the nitrated residue was not a tyrosine but a tryptophan located at 372. These diverse conclusions prompted us to perform a systematic investigation into the nitration of SCOT, how the 10.1021/pr100349g
2010 American Chemical Society
Characterization of Nitrated Tyrosine Residues in SCOT catalytic changes of SCOT correspond to nitration of the specific site(s). In this communication, we report a functional proteomic investigation, in which nitrated mitochondrial proteins in the db/db diabetic mouse heart were first surveyed using 2DEWestern blot with 3NT antibody. We found mitochondrial proteins heavily nitrated due to diabetic development and identified 18 nitrated proteins that were mitochondria-specific and related to energy metabolism. Of these nitrated mitochondrial proteins, SCOT is a typical target of nitration so that it was selected for exploring the nitrated site(s) and the corresponding changes of catalysis. With LC-ESI-MS/MS, nitrated residues in the recombinant SCOT were identified at Tyr4 and Tyr76. This conclusion was further confirmed by site-directed mutagenesis and specific catalytic activity. For the first time, we gained the solid evidence that Tyr4 and Tyr76 of SCOT are the priority sites attacked by ONOO-, and nitration of the two tyrosine residues is a causative factor of SCOT inactivation.
Experimental Procedures The Diabetic Mouse Model. The C57BLKS/J db/db mice, generated from Jackson Laboratory, were kindly donated by Dr. Guan’s laboratory in Peking University Diabetes Center.23 The procedures of animal treatment were in accordance with recommendations published in the Guide for the Care and Use of Laboratory Animals. The mice were housed under a 12 h/12 h light-dark cycle with access to standard rodent chow. Mouse bodies were weighed and blood glucose concentrations were measured. After 14-16 weeks, the diabetic (db/db) or control (db/+m) mice were sacrificed, and the freshly isolated hearts were immediately delivered to mitochondrial preparation. Mitochondrial Preparation. Briefly, fresh hearts from mice were minced with scissors and homogenized in a buffer containing 0.25 M sucrose, 0.5% protease inhibitor cocktail (Sigma), 0.5 mM EGTA, and 10 mM HEPES, pH 7.5, with a glass homogenizer. After centrifugation at 1000g to remove the debris, the supernatants were centrifuged at 10 000g for 30 min and the resulting pellets were the raw preparation of mitochondria, which were used in SCOT catalytic activity assays and Western blots. Protein Separation by Two-Dimensional Electrophoresis (2DE). Mitochondrial proteins were extracted by a lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 10 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM EDTA, and 40 mM Tris-HCl, pH 7.4. The prepared proteins were loaded onto 18 cm, linear pH 3-10 IPG strips and rehydrated in 40 mM Tris-HCl, pH 7.4, 8 M urea, 2% (w/v) CHAPS, 65 mM DTT, and 0.5% IPGphor buffer. The focused strips were treated by reduction with 1% (w/v) DTT and alkylation with 2.5% (w/v) iodoacetamide (IAM) in equilibration buffer containing 50 mM Tris-HCL, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and trace Bromophenol blue. The treated strips were transferred onto 24 cm 12% acrylamide SDS-PAGE gel. The 2DE gels were stained by silver nitrate according to the protocol from Amersham only without glutardialdehyde. All the silver-stained gels were scanned by a laser densitometer at 500 pixel resolution (Powerlook 2100XL, UMAX, Dallas, TX). The gel images were analyzed with Imagemaster Platinum version 5.0 (GE Healthcare, Fairfield, CT). Western Blot. Protein samples derived from the recombinants or mitochondria were first electrophoresed by SDS-PAGE or 2DE and then electrotransferred onto PVDF membranes by a Bio-Rad Mini PROTEAN 3 system. The polyclonal anti-SCOT
technical notes and anti-HADHB antibodies were generated from our laboratory by immunization of rabbits with the purified SCOT and HADHB recombinant protein; the monoclonal anti-3NT and anti-ATP synthase β antibodies were purchased from Calbiochem and BD Biosciences, respectively; and anti-iNOS and anti-sMtCK polyclonal antibodies were obtained from Santa Cruz. A secondary antibody conjugated with horseradish peroxidase was used to amplify the immuno-recognition signals with the peroxidase activity monitored using an ECL kit (GE Healthcare). For 2DE-Western blot, the mitochondrial proteins were first separated by isoelectric focusing on 13 cm IPG strip with linear pH 3-10, and then transferred onto 13 cm 12% acrylamide SDS-PAGE gel for the second-dimensional separation. Generally, a mitochondrial sample was divided for two parallel runs for 2DE. After 2DE, one gel was directly stained by silver staining, and the other further electrotransferred onto PVDF membranes. The nitrated proteins on PVDF were recognized by 3NT antibody followed by ECL staining. To elucidate which spot has the positive immune-reactive signals, the ECL films of 2DE-Western blot were labeled with alignment marks and carefully overall superposed with the silver gels. The silver staining spots corresponding to the immune-positive ones were excised and delivered toward for protein identification. Protein Identification by Mass Spectrometry. On the basis of the 2DE-Western blot image analysis, immuno-reactive protein spots on the 2DE gel were excised, reduced, alkylated, and digested with trypsin. The digested products were loaded onto Anchorchip target (Bruker Dalton, Bremen, Germany) and analyzed on an Ultraflex TOF/TOF mass spectrometer (Bruker Dalton, Bremen, Germany) for protein identification. Typically, 100 shots were accumulated per spectrum in MS mode and 400 shots in MS/MS mode. The spectra were processed using the FlexAnalysis 2.2 and BioTools 2.2 software tools. Protein identification was performed using the Mascot software (http:// www.matrixscience.com) to search the NCBInr database with mouse as taxonomy. The following parameters were used for database searches: Monoisotopic mass accuracy
